SCIENCE CHINA Life Sciences, Volume 61 , Issue 9 : 1030-1038(2018) https://doi.org/10.1007/s11427-017-9290-1

Loss of Hox5 function results in myofibroblast mislocalization and distal lung matrix defects during postnatal development

More info
  • ReceivedFeb 20, 2018
  • AcceptedMar 23, 2018
  • PublishedApr 27, 2018


Alveologenesis is the final stage of lung development and is responsible for the formation of the principle gas exchange units called alveoli. The lung mesenchyme, in particular the alveolar myofibroblasts, are drivers of alveolar development, however, few key regulators that govern the proper distribution and behavior of these cells in the distal lung during alveologenesis have been identified. While Hox5 triple mutants (Hox5 aabbcc) exhibit neonatal lethality, four-allele, compound mutant mice (Hox5 AabbCc) are born in Mendelian ratios and are phenotypically normal at birth. However, they exhibit defects in alveologenesis characterized by a BPD-like phenotype by early postnatal stages that becomes more pronounced at adult stages. Invasive pulmonary functional analyses demonstrate significant increases in total lung volume and compliance and a decrease in elastance in Hox5 compound mutants. SMA+ myofibroblasts in the distal lung are distributed abnormally during peak stages of alveologenesis and aggregate, resulting in the formation of a disrupted elastin network. Examination of other key components of the distal lung ECM, as well as other epithelial cells and lipofibroblasts reveal no differences in distribution. Collectively, these data indicate that Hox5 genes play a critical role in alveolar development by governing the proper cellular behavior of myofibroblasts during alveologenesis.

Funded by

a Ruth L. Kirschstein National Research Service Award(NSRA)


and Blood Institute(NHLBI)


This work was supported by a Ruth L. Kirschstein National Research Service Award (NSRA) training Grant 5 T32 HL 7749-20 to S.M.H. This research was also supported by MICHR PTSP UL1TR002240 to L.M.S and the National Heart, Lung, and Blood Institute (NHLBI) R01-HL119215 to D.M.W.

Interest statement

The author(s) declare that they have no conflict of interest.



Table S1 Primer sequences used

Figure S1 Hox5 AabbCc compound mutants have normal trachea at E18.5. Skeletal preparations of compound Hox5 AabbCc mutant mice reveal no phenotypic changes of the trachea (A) compared to the clear tracheal defects observed in Hox5 aaBBCC single mutants (B) at E18.5. Scale bar represents 1.0 mm.

Figure S2 qPCR of ECM components in distal airway of Hox5 AabbCc compound mutants at P7. qPCR measures no difference in the expression levels of several laminin mRNAs, Col4a1 and Col4a2, Col3a1, and fibronectin in Hox5 AabbCc mutants at P7. n=3 control and Hox5 AabbCc mutant animals were measured in qPCR analyses.

Supplemental movie1 (S1) 3D reconstruction of α-SMA myofibroblasts (red) in the distal lung region of control animals at P7.

Supplemental movie2 (S2) 3D reconstruction of α-SMA myofibroblasts (red) in the distal lung region of Hox5 AabbCc mutants animals at P7.

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


[1] Ahlfeld S.K., Conway S.J.. Aberrant signaling pathways of the lung mesenchyme and their contributions to the pathogenesis of bronchopulmonary dysplasia. Birth Defects Res Part A-Clinical Mol Teratology, 2012, 94: 3-15 CrossRef PubMed Google Scholar

[2] Aubin J., Lemieux M., Tremblay M., Bérard J., Jeannotte L.. Early postnatal lethality in Hoxa-5 mutant mice is attributable to respiratory tract defects. Dev Biol, 1997, 192: 432-445 CrossRef PubMed Google Scholar

[3] Boucherat O., Montaron S., Bérubé-Simard F.A., Aubin J., Philippidou P., Wellik D.M., Dasen J.S., Jeannotte L.. Partial functional redundancy betweenHoxa5 andHoxb5 paralog genes during lung morphogenesis. Am J Physiol-Lung Cellular Mol Physiol, 2013, 304: L817-L830 CrossRef PubMed Google Scholar

[4] Branchfield K., Li R., Lungova V., Verheyden J.M., McCulley D., Sun X.. A three-dimensional study of alveologenesis in mouse lung. Dev Biol, 2016, 409: 429-441 CrossRef PubMed Google Scholar

[5] Brody, J.S., and Kaplan, N.B. (1983). Proliferation of alveolar interstitial cells during postnatal lung growth. Evidence for two distinct populations of pulmonary fibroblasts. Am Rev Resp Dis 127, 763-770. Google Scholar

[6] Butts T., Holland P.W.H., Ferrier D.E.K.. The urbilaterian Super-Hox cluster. Trends Genets, 2008, 24: 259-262 CrossRef PubMed Google Scholar

[7] Condie B.G., Capecchi M.R.. Mice with targeted disruptions in the paralogous genes hoxa-3 and hoxd-3 reveal synergistic interactions. Nature, 1994, 370: 304-307 CrossRef PubMed ADS Google Scholar

[8] Di Meglio T., Kratochwil C.F., Vilain N., Loche A., Vitobello A., Yonehara K., Hrycaj S.M., Roska B., Peters A.H.F.M., Eichmann A., et al. Ezh2 orchestrates topographic migration and connectivity of mouse precerebellar neurons. Science, 2013, 339: 204-207 CrossRef PubMed ADS Google Scholar

[9] Duboule, D. (1994). Temporal colinearity and the phylotypic progression: a basis for the stability of a vertebrate Bauplan and the evolution of morphologies through heterochrony. Dev Suppl, 135-142. Google Scholar

[10] Duboule, D., and Dolle, P. (1989). The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes. Embo J 8, 1497-1505. Google Scholar

[11] Galambos C., Demello D.E.. Regulation of alveologenesis clinical implications of impaired growth. Pathology, 2008, 40: 124-140 CrossRef PubMed Google Scholar

[12] Graham A., Papalopulu N., Krumlauf R.. The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell, 1989, 57: 367-378 CrossRef Google Scholar

[13] Hamilton T.G., Klinghoffer R.A., Corrin P.D., Soriano P.. Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol Cellular Biol, 2003, 23: 4013-4025 CrossRef Google Scholar

[14] Hantos Z., Adamicza A., Jánosi T.Z., Szabari M.V., Tolnai J., Suki B.. Lung volumes and respiratory mechanics in elastase-induced emphysema in mice. J Appl Physiol, 2008, 105: 1864-1872 CrossRef PubMed Google Scholar

[15] Hines E.A., Sun X.. Tissue crosstalk in lung development. J Cell Biochem, 2014, 115: 1469-1477 CrossRef PubMed Google Scholar

[16] Holland P.W.H.. Evolution of homeobox genes. WIREs Dev Biol, 2013, 2: 31-45 CrossRef PubMed Google Scholar

[17] Holland, P.W. (2015). Did homeobox gene duplications contribute to the Cambrian explosion? Zool Let 1, 1. Google Scholar

[18] Horan G.S., Ramirez-Solis R., Featherstone M.S., Wolgemuth D.J., Bradley A., Behringer R.R.. Compound mutants for the paralogous hoxa-4, hoxb-4, and hoxd-4 genes show more complete homeotic transformations and a dose-dependent increase in the number of vertebrae transformed.. Genes Dev, 1995, 9: 1667-1677 CrossRef Google Scholar

[19] Hrycaj S.M., Dye B.R., Baker N.C., Larsen B.M., Burke A.C., Spence J.R., Wellik D.M.. Hox5 genes regulate the Wnt2/2b-Bmp4-signaling axis during lung development. Cell Rep, 2015, 12: 903-912 CrossRef PubMed Google Scholar

[20] Hsieh-Li, H.M., Witte, D.P., Weinstein, M., Branford, W., Li, H., Small, K., and Potter, S.S. (1995). Hoxa 11 structure, extensive antisense transcription, and function in male and female fertility. Development 121, 1373-1385. Google Scholar

[21] Kessel M., Gruss P.. Murine developmental control genes. Science, 1990, 249: 374-379 CrossRef ADS Google Scholar

[22] Klinghoffer R.A., Hamilton T.G., Hoch R., Soriano P.. An allelic series at the PDGFαR locus indicates unequal contributions of distinct signaling pathways during development. Dev Cell, 2002, 2: 103-113 CrossRef Google Scholar

[23] Krumlauf R.. Evolution of the vertebrateHox homeobox genes. BioEssays, 1992, 14: 245-252 CrossRef PubMed Google Scholar

[24] Krumlauf, R. (1993). Mouse Hox genetic functions. Curr Opin Genet Dev 3, 621-625. Google Scholar

[25] Larsen B.M., Hrycaj S.M., Newman M., Li Y., Wellik D.M.. MesenchymalHox6 function is required for mouse pancreatic endocrine cell differentiation. Development, 2015, 142: 3859-3868 CrossRef PubMed Google Scholar

[26] Leucht P., Kim J.B., Amasha R., James A.W., Girod S., Helms J.A.. Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration. Development, 2008, 135: 2845-2854 CrossRef PubMed Google Scholar

[27] Mandeville I., Aubin J., LeBlanc M., Lalancette-Hébert M., Janelle M.F., Tremblay G.M., Jeannotte L.. Impact of the loss of Hoxa5 function on lung alveogenesis. Am J Pathol, 2006, 169: 1312-1327 CrossRef PubMed Google Scholar

[28] Manley, N.R., and Capecchi, M.R. (1995). The role of Hoxa-3 in mouse thymus and thyroid development. Development 121, 1989-2003. Google Scholar

[29] Manley N.R., Capecchi M.R.. HoxGroup 3 paralogs regulate the development and migration of the thymus, thyroid, and parathyroid glands. Dev Biol, 1998, 195: 1-15 CrossRef PubMed Google Scholar

[30] Martinez, P., and Amemiya, C.T. (2002). Genomics of the HOX gene cluster. Comp Biochem Phys B 133, 571-580. Google Scholar

[31] McCulley, D., Wienhold, M., and Sun, X. (2015). The pulmonary mesenchyme directs lung development. Curr Opin Genet Dev 32, 98-105. Google Scholar

[32] McGinnis W., Krumlauf R.. Homeobox genes and axial patterning. Cell, 1992, 68: 283-302 CrossRef Google Scholar

[33] McIntyre D.C., Rakshit S., Yallowitz A.R., Loken L., Jeannotte L., Capecchi M.R., Wellik D.M.. Hox patterning of the vertebrate rib cage. Development, 2007, 134: 2981-2989 CrossRef PubMed Google Scholar

[34] Morrisey E.E., Hogan B.L.M.. Preparing for the first breath: genetic and cellular mechanisms in lung development. Dev Cell, 2010, 18: 8-23 CrossRef PubMed Google Scholar

[35] Papagiannouli F., Schardt L., Grajcarek J., Ha N., Lohmann I.. The Hox gene Abd-B controls stem cell niche function in the Drosophila testis. Dev Cell, 2014, 28: 189-202 CrossRef PubMed Google Scholar

[36] Pineault K.M., Swinehart I.T., Garthus K.N., Ho E., Yao Q., Schipani E., Kozloff K.M., Wellik D.M.. Hox11 genes regulate postnatal longitudinal bone growth and growth plate proliferation. Biol Open, 2015, 4: 1538-1548 CrossRef PubMed Google Scholar

[37] Ptaschinski C., Hrycaj S.M., Schaller M.A., Wellik D.M., Lukacs N.W.. Hox5 paralogous genes modulate Th2 cell function during chronic allergic inflammation via regulation ofGata3. JI, 2017, 199: 501-509 CrossRef PubMed Google Scholar

[38] Rinn J.L., Wang J.K., Allen N., Brugmann S.A., Mikels A.J., Liu H., Ridky T.W., Stadler H.S., Nusse R., Helms J.A., et al. A dermal HOX transcriptional program regulates site-specific epidermal fate. Genes Dev, 2008, 22: 303-307 CrossRef PubMed Google Scholar

[39] Rousso D.L., Gaber Z.B., Wellik D., Morrisey E.E., Novitch B.G.. Coordinated actions of the forkhead protein Foxp1 and Hox proteins in the columnar organization of spinal motor neurons. Neuron, 2008, 59: 226-240 CrossRef PubMed Google Scholar

[40] Rux D.R., Song J.Y., Pineault K.M., Mandair G.S., Swinehart I.T., Schlientz A.J., Garthus K.N., Goldstein S.A., Kozloff K.M., Wellik D.M.. Hox11 function is required for region-specific fracture repair. J Bone Miner Res, 2017, 32: 1750-1760 CrossRef PubMed Google Scholar

[41] Rux D.R., Song J.Y., Swinehart I.T., Pineault K.M., Schlientz A.J., Trulik K.G., Goldstein S.A., Kozloff K.M., Lucas D., Wellik D.M.. Regionally restricted Hox function in adult bone marrow multipotent mesenchymal stem/stromal cells. Dev Cell, 2016, 39: 653-666 CrossRef PubMed Google Scholar

[42] Sajjan U., Ganesan S., Comstock A.T., Shim J., Wang Q., Nagarkar D.R., Zhao Y., Goldsmith A.M., Sonstein J., Linn M.J., et al. Elastase- and LPS-exposed mice display altered responses to rhinovirus infection. Am J Physiol-Lung Cell Mol Physiol, 2009, 297: L931-L944 CrossRef PubMed Google Scholar

[43] Schughart, K., Kappen, C., and Ruddle, F.H. (1988). Mammalian homeobox-containing genes: genome organization, structure, expression and evolution. Br J Cancer Suppl 9, 9-13. Google Scholar

[44] Stultz B.G., Park S.Y., Mortin M.A., Kennison J.A., Hursh D.A.. Hox proteins coordinate peripodial decapentaplegic expression to direct adult head morphogenesis in Drosophila. Dev Biol, 2012, 369: 362-376 CrossRef PubMed Google Scholar

[45] Vanoirbeek J.A.J., Rinaldi M., De Vooght V., Haenen S., Bobic S., Gayan-Ramirez G., Hoet P.H.M., Verbeken E., Decramer M., Nemery B., et al. Noninvasive and invasive pulmonary function in mouse models of obstructive and restrictive respiratory diseases. Am J Respir Cell Mol Biol, 2010, 42: 96-104 CrossRef PubMed Google Scholar

[46] Wellik D.M.. Hox patterning of the vertebrate axial skeleton. Dev Dyn, 2007, 236: 2454-2463 CrossRef PubMed Google Scholar

[47] Wellik, D.M. (2009). Hox genes and vertebrate axial pattern. Curr Top Dev Biol 88, 257-278. Google Scholar

[48] Wellik D.M., Hawkes P.J., Capecchi M.R.. Hox11 paralogous genes are essential for metanephric kidney induction. Genes Dev, 2002, 16: 1423-1432 CrossRef PubMed Google Scholar

[49] Xu B., Hrycaj S.M., McIntyre D.C., Baker N.C., Takeuchi J.K., Jeannotte L., Gaber Z.B., Novitch B.G., Wellik D.M.. Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb. Proc Natl Acad Sci USA, 2013, 110: 19438-19443 CrossRef PubMed ADS Google Scholar

[50] Yallowitz A.R., Hrycaj S.M., Short K.M., Smyth I.M., Wellik D.M.. Hox10 genes function in kidney development in the differentiation and integration of the cortical stroma. PLoS ONE, 2011, 6: e23410 CrossRef PubMed ADS Google Scholar

  • Figure 1

    Hox5 four-allele compound mutants (Hox5 AabbCc) exhibit alveolar simplification during early postnatal and adult stages. Newborn Hox5 compound mutants are histologically indistinguishable from controls and have no measurable difference in alveolar chord length (A–B’; G). Scale bars in B, B’ represent 200 and 100 µm, respectively. By two weeks of age, Hox5 compound mutants exhibit enlarged, simplified alveoli compared to controls with a statistically significant ~33% increase in alveolar chord length (C–D’; G). Scale bars in C, C’ represent 200 and 100 µm, respectively. The most severe alveolar simplification phenotype of Hox5 AabbCc mutants occurs at adult stages, evidenced by the greatest increase in alveolar chord length (~100%) compared to controls (E–F’; G). Scale bars in F, F’ represent 400 and 100 µm, respectively. n=4 animals were measured for both control and Hox5 AabbCc groups for each stage depicted. P values and statistical significance (*) were determined by an unpaired Student’s t test.

  • Figure 2

    Hox5 AabbCc adults exhibit abnormal pulmonary function. Quasistatic and fast flow maneuvers were performed using the Buxco system to measure lung volume, compliance and elastance in tracheotomized mice. Increased lung volumes of Hox5 AabbCc mice are indicated by significant increases in inspiratory capacity (IC) (A), vital capacity (VC) (B), forced vital capacity (FVC) (C) and forced expiratory volume (FEV) (D). Increased lung compliance of Hox5 AabbCc mutants are determined by significant increases in chord compliance (Cchord) (E), compliance at zero pressure (Cp0) (F) and peak compliance (Cpk) (G). Hox5 AabbCc animals also exhibit a significant decrease in elastance at adult stages (H). n=4 control and Hox5 AabbCc animals were measured for PFT analyses. P values and statistical significance (*) were determined by an unpaired Student’s t test.

  • Figure 3

    Major ECM components of the distal airway are unaffected in Hox5 AabbCc mutants at P7. IHC analyses reveal no changes in the protein expression patterns of basement membrane components laminin (A, B) and Col4 (C, D) in Hox5 AabbCc mutants compared to controls. We also see no differences in the expression patterns of Col3 (E, F) or fibronectin (G, H) in the distal airway of Hox5 compound mutants at P7. Scale bars represent 50 µm.

  • Figure 4

    The elastin-based network is perturbed in Hox5 AabbCc mutants at P7. The elastin network forms tightly organized bundles that are localized around the alveolar openings and septal tips in control animals at P7 (A, A’). In contrast, the elastin network in Hox5 compound mutants is highly disorganized with entangled elastin fibers within the thickened lung parenchyma in the distal airway (B, B’). Scale bars in B, B’ represent 100 and 50 µm, respectively.

  • Figure 5

    Alveolar myofibroblasts are abnormally localized in the distal airway of Hox5 AabbCc mutants at P7. Control animals show a highly organized network of interconnected SMA+ alveolar myofibroblasts at P7 that are relatively evenly distributed around the alveolar openings and septal tips (A). In contrast, the network of SMA+ alveolar myofibroblasts is markedly more diffuse in Hox5 AabbCc mutants compared to controls with abnormal distribution and aggregation of these cells within the thickened lung parenchyma (A). Scale bars in (A) represent 200, 100 and 50 µm (left to right). SMA+ alveolar myofibroblasts continue to be tightly associated with elastin in Hox5 AabbCc mutants at P7, even in the distal lung regions that exhibit parenchymal thickening and myofibroblast clumping (B). Scale bar in (B) represents 50 µm. As observed in control animals, all SMA+ myofibroblasts co-express PdgfrαGFP in Hox5 AabbCc mutants and quantification reveals no difference in the total number of double positive cells at P7 (C). Scale bars in (C) represent 100 and 50 µm (left to right). n=3 control and Hox5 AabbCc mutant animals were used for quantification analyses.

  • Figure 6

    Additional cell types of the distal lung airway are unaffected in Hox5 AabbCc mutants at P7. IHC analyses reveal no differences in the distribution or morphology of T1α+ AECI cells (A, B) or Sftpc+ AECII cells (C, D) in Hox5 compound mutants. Adrp+ lipofibroblasts are normally distributed in the distal airway of Hox5 AabbCc mutants (E, F). Scale bars represent 50 µm. qPCR measures no differences in the expression levels of Elastin, T1α, Sftpc or Adrp in Hox5 compound mutants at P7. n=3 control and Hox5 AabbCc mutant animals were measured in qPCR analyses.

Copyright 2020 Science China Press Co., Ltd. 《中国科学》杂志社有限责任公司 版权所有

京ICP备17057255号       京公网安备11010102003388号